There have been significant advances in recent years in the development of microfluidic/lab-on-a-chip (LOC) cellular assays, with numerous papers detailing different detection/analysis methods which may be used in such devices [1-5]. However, these all tend to be interrogation methods which provide measurements of specific parameters, e.g. a change in intensity or lifetime of a fluorophore. A more general monitoring device would find wider application. At present all optical imaging of biological samples, with a few exceptions, is still undertaken on large expensive microscopes that obviously do not exploit many of the advantages offered by LOC devices, such as small size and low cost.
One notable exception is the optofluidic microscope (OFM) [6] developed by Heng et al., which has a resolution comparable to that offered by conventional microscopy (measured to be 490+/−40 nm). This method however, relies on the sample being moved across the sensor at a known velocity making the OFM unsuitable in situations where the cell/sample/object of interest remains fixed or moves at an unknown velocity, such as in clonogenic or chemotaxis type assays.
Lange et al. developed a shadow imager [7] for studying the effects of space flight on nematode Caenorhabditis elegans. In this system C. elegans are placed directly on top of an area imager and illuminated with collimated light, thereby casting a shadow onto the sensor. The resolution of the resultant image is inherently set by the pitch and pixel size of the video camera chip, making (in practice) the detection of single cells (e.g. mammalian cells with a diameter of =15 μm) extremely difficult if not impossible. Also most cells are transparent at visible wavelengths and thus may not produce a discernable shadow.
Current technologies for monitoring live cells over time are largely reliant on manual intervention and expertise; automatic technologies are immature or bulky and expensive.
Any application where cells have to be kept alive and viable for extended periods of time (several days to weeks) requires the use of an incubator. Large, laboratory-based incubators are generally used, and these maintain a suitable temperature and gas mixture environment. Monitoring of the cells must be performed on a microscope, which requires an operator to transfer the dish or flask of cells from the incubator. A trained operator usually then views the cells using phase-contrast microscopy.
Automation of this process may be performed by the creation of an incubation chamber that surrounds the microscope, but keeping cells viable in this environment is usually more problematic. This approach also requires the use of an expensive automated microscope. Detection and tracking of the cells by processing the images from the phase-contrast microscope is also possible in principle, but is difficult in practice because of the large variability in the visual appearance of cells when visualized in this manner. One attempt to automate and miniaturize this type of system is the CellQ product (Chip-Man Technologies Ltd, Finland).
The use of point-source illumination with a coherent source (e.g. laser) has been described previously [13, 14, 15], but with the intention to record holographic information. In such approaches, some form of reconstruction technique must be employed; recent approaches have relied on numerical or computational methods to perform the reconstruction. Furthermore, such approaches require the source diameter to be comparable or smaller than the wavelength of light used.
In some cases, Fraunhofer diffraction has been used to establish the power going into certain spatial frequencies by measurement of the relative brightness of an annular region [16]. In this instance, a laser source is used and only one or few objects are imaged.